Our research focuses on elucidating new fundamental principles governing T cell activation, regulation, and effector function, and employing these to develop more effective vaccine and immunotherapy strategies for HIV and cancer. This involves several steps that together comprise a push-pull approach. First, we optimize the antigen to improve immunogenicity by epitope enhancement, changing the amino acid sequence to increase affinity for the relevant major histocompatibility (MHC) molecule. We have done this for several cancer antigens, including 2 new prostate cancer antigens, TARP and POTE, and 3/4-accrued a phase I/II TARP clinical trial in D0 prostate cancer patients with rising PSA levels. The slope of PSA rise has significantly decreased among the first 29 patients enrolled (p = 0.045 pre to 24 wks;p = 0.027 pre to 48 wks), suggesting slowing of cancer growth. The second step is to push the response with molecular adjuvants, such as cytokines and Toll-like receptor (TLR) ligands, to improve not only the quantity but also the quality of the response. We published that IL-15 is an important mediator of CD4 T cell help for CD8 T cells, being sufficient to substitute for help in animals depleted of CD4 T cells, to promote CD8 longevity and prevent TRAIL-mediated apoptosis, and also necessary for optimal help. We also previously found that IL-15 increased the avidity of the CD8 T cells, necessary for effective clearance of virus or tumor cells. We translated this to humans showing that IL-15 could substitute for CD4 help to induce a primary in vitro CD8 T cell response of naive T cells whereas IL-2 could not. IL-15 also restored alloresponsiveness of CD4 T cells from HIV-infected patients to normal levels. We also found that IL-1beta as adjuvant could enhance CD8 T cell responses and skew CD4 help to Th17. We also investigated TLR ligands as adjuvants, as these can mature DCs and induce their production of cytokines like IL-12 and IL-15. We showed synergy between pairs of TLR ligands that work through different intracellular signal transducers, MyD88 or TRIF, and determined the mechanism in DCs involving unidirectional cross-talk from TRIF to enhance MyD88-dependent cytokine production. We have now published that a triple TLR ligand combination that induces more effective protection against virus infection does not increase T cell quantity, but improves quality by inducing higher avidity T cells, and more IL-15 production. We tested the combination of triple TLR ligands, IL-15, both or neither as vaccine adjuvants in a peptide-prime, MVA-boost mucosal vaccine for SIV in macaques, challenging intrarectally with SIVmac251. As only macaques receiving both types of adjuvants showed partial protection, we investigated correlates of protection. In the adaptive immune arm, surprisingly only polyfunctional CD8 T cells specific for SIV antigens, but not total specific T cells measured by peptide-MHC tetramer binding, correlated with protection. In the innate immune arm, we found the adjuvants induced long-lived innate protection by APOBEC3G. These adjuvants also increased CD4 cell preservation in the gut, independent of viral load. Thus, molecular adjuvant vaccine strategies inducing both innate and adaptive immunity may be the most efficacious. The third step is to target the immune response to the relevant tissue, the mucosa in the case of HIV. We published a study of mucosal T cell trafficking in which we discovered a lack of equilibrium between T cells populating the intraepithelial compartment and the lamina propria in the small intestine, leading to a distinct founder effect. We also found that homing to the large intestine is governed in part by DCs from colon patches, using a mechanism independent of retinoic acid, distinct from that in the small intestine. We are identifying chemokines to selectively target T cells to the large intestine. We also developed a novel nanoparticle approach to vaccine delivery to the large intestine, using vaccine nanoparticles coated with Eudragit FS30D to allow oral delivery and release of the particles primarily in the large intestine, bypassing the stomach and site of oral tolerance in the small intestine. This could substitute for intrarectal delivery to induce protection against viral challenge via the rectal or vaginal route. We have recently adapted the oral nanoparticle approach to non-human primates in an AIDS vaccine. The fourth step is to pull the response by removing the brakes, i.e., blocking the negative regulatory mechanisms that inhibit immunity. We previously discovered a new immunoregulatory pathway involving NKT cell suppression of tumor immunity. The NKT cells make IL-13 that induces myeloid cells to make TGF-beta, which suppresses the CD8 T cell response. As NKT cells can also protect against tumors, we needed to resolve this paradox. We found that type I NKT cells (using an invariant TCRalpha chain) protected, whereas type II NKT cells (using diverse TCRs) suppressed immunity. Moreover, selective activation of type I or type II NKT cells showed they cross-regulated each other, defining a new immunoregulatory axis, analogous to the axis between Th1 and Th2 cells that has profoundly affected immunology. The balance along the NKT axis could influence subsequent adaptive immune responses. We found that type II NKT cells also suppress conventional CD4 and CD8 antigen-specific T cells. We are researching tumor lipids that stimulate NKT cells, markers to identify type II NKT cells, the mechanisms of suppression and also the relationship between suppressive NKT cells and CD25+ Foxp3+ T regulatory cells. We found that when type I NKT cells control type II, T reg cells dominate, but loss of type I NKT cells can reveal suppression by type II NKT cells. Conversely, stimulating with a type I NKT cell agonist can protect against tumors. We discovered that a beta-mannosyl analog of alpha-galactosylceramide, in contrast to other beta-linked sugar ceramides, is also protective. However, its mechanism of protection against cancer is different from that of the classic alpha-GalCer, being dependent on TNF-alpha and nitric oxide synthase rather than on interferon-gamma, and it synergizes with alpha-GalCer. It also does not induce the degree of anergy found after alpha-GalCer injection. It also stimulates human NKT cells. This first representative of a new class of NKT cell agonists should be translated to human cancer therapy. A key mediator of the NKT regulatory pathway and an important regulator of T regulatory cells is TGFbeta. We found that blockade of TGFbeta can protect against certain tumors in mice, and can synergize with anti-cancer vaccines in 2 mouse models. The protection is dependent on CD8 T cells, and in combination with a vaccine, the anti-TGFbeta increases the number of both total and high avidity CD8 T cells. We have translated this into a human clinical trial of a human anti-TGFbeta monoclonal antibody in a CRADA with Genzyme, in melanoma and renal cell cancer. The phase I study showed some activity, paradoxically mostly at lower doses (one long partial remission, 3 mixed responses and 2 cases of stable disease among 22 patients). We are obtaining regulatory approval for a phase II trial in melanoma to compare a low and high dose of antibody. Finally, we recently found that an adenovirus vaccine expressing the extracellular and transmembrane domains of HER-2 can cure large established mammary cancers and lung metastases in mice. The mechanism surprisingly involves antibodies that inhibit HER-2 function, rather than T cells. We have now made a similar cGMP recombinant adenovirus expressing the human HER-2 domains to carry out a clinical trial in human cancer patients and are submitting an IND.